IL-2 is a soluble protein which is capable of modulating lymphocyte activation (mitogenesis) and, in the past, has been produced by stimulating mouse, rat or human lymphocyte cells with a mitogen. For instance, Morgan et al. in "Selective in vitro Growth of T Lymphocytes from Normal Human Bone Marrow", 193 Science 1007 (1976) and Ruscetti et al., in "Functional and Morphological Characterization of Human T Cells Continuously Grown in vitro", 119 The Journal of Immunology 131 (1977), both discussed a process for culturing pooled normal human lymphocytes in Roswell Park Memorial Institute (hereafter "RPMI") medium containing autologous serum and the mitogen, phytohemagglutinin (hereafter "PHA").
Gillis and Smith, in "Long Term Culture of Tumor-Specific Cytotoxic T Cells", 268 Nature 154 (1977), reported preparing murine IL-2 by stimulating with the mitogen concanavalin A (hereafter "Con A"), a culture medium composed of normal DBA/2 spleen cells conditioned with RPMI 1640 and fetal calf serum (hereafter "FCS")), and Farrar et al. in "Biological Relationship of Thymocyte Mitogenic Factor and Factors Enhancing Humoral and Cell-Mediated Immune Responses", 121 The Journal of Immunology 1353 (1978), disclosed preparing IL-2 from normal murine spleen cells incubated with Con A in a tissue culute medium containing normal mouse serum (hereafter "NMS").
Gillis et al. reported generating IL-2 from normal murine and rat spleen cells cultured in a medium consisting of RPMI 1640 tissue culture medium supplemented with heat-inactivated FCS, penicillin-G, and gentamycin in a humidified atmosphere of CO.sub.2 and air. The murine and rat spleen cells were stimulated by various mitogens including Con A, PHA and pokeweed mitogen (hereafter "PKM"). "Cell Growth Factor: Parameters of Production and a Quantative Microassay for Activity", 120 The Journal of Immunology 2027 (1978).
Gillis et al. in "Biochemical and Biological Characterization of Lymphocyte Regulatory Molecules V. Identification of an Interleukin-2 Producing Human Leukemia T Cell Line", 152 The Journal of Experimental Medicine 1709 (1980), identified the preparation of IL-2 from a human malignant T cell line, specifically malignant leukemic T cells (Jurkat-FHCRC) cultured in RPMI 1640 supplemented with heat-inactivated FCS, N-2-hydroxy-piperazine-XI.sup.1 -2-ethene-sidfonic acid (hereafter "Hepes") buffer, penicillin, streptomycin, NaHCO.sub.3 and fresh L-glutamine. The cultures were stimulated with various mitogens including Con A, PHA and PKM.
IL-2 purified from these mouse, rat and human cell lines has been found to retain different types of biological activity, including: (1) marked enhancement of thymocyte mitogenesis, Farrer et al., supra, 121 The Journal of Immunology 1352; (2) promotion of long term in vitro proliferation of antigen specific helper or killer T cell lines, Gillis and Smith, supra, 268 Nature 154, and Watson, "Continuous Proliferation of Murine Antigen Specific Helper T Lymphocytes in Culture", 150 Journal of Experimental Medicine 1510 (1979); (3) induction of cytotoxic T lymphocyte (hereafter "CTL") generation in both alloantigen-stimulated thymocyte and nude spleen cell cultures, Watson et al., "Biochemical and Biological Characterization of Lymphocyte Regulatory Molecules I. Purification of a Class of Murine Lymphokines", 150 Journal of Experimental Medicine 849 (1979), and Gillis et al., "Biochemical and Biological Characterization of Lymphocyte Regulatory Molecules II. Purification of a Class of Rat and Human Lymphokines", 124 The Journal of Immunology 1954 (1980); and (4 ) promotion of anti-erythrocyte, (Red Blood Cell) plaque-forming cell responses in nude spleen cell cultures stimulated with sheep red blood cells, Watson et al., supra, 150 Journal of Experimental Medicine 849, and Gillis et al, supra, 124 The Journal of Immunology 1954. Accordingly, these identified biological activities of IL-2 in immune response assays indicate that murine IL-2 is useful in the study of immune responses in various diseases, such as immune deficient or neoplastic diseases.
The above cited articles by Gillis & Smith, 268 Nature 154; and Farrar et al., 121 The Journal of Immunology 2027, discuss production of murine IL-2 from lectin stimulated normal spleen cells. However, current production sources and techniques result in very weak concentrations of IL-2. Fractionation of large volumes of conventionally prepared conditioned media containing IL-2 results in only very small quantities of purified murine IL-2. As a consequence, sufficient quantities of concentrated murine IL-2 have not been available for in vivo experiments nor have been available to study the finite molecular character of this lymphocyte regulatory molecule. Accordingly, a principal object of the present invention is to identify cell lines and clones thereof which are potent producers of murine IL-2 and also to ascertain the particular conditions and specific mitogens which foster optimum production of murine IL-2 from such identified cell lines and cell line clones.